Antitumor agents

ABSTRACT

Novel mono-, di- and triacylated derivatives of scirpentriol are provided for use as antitumor agents. Also provided are methods for the production of such derivatives.

This application is a division of our co-pending application Ser. No. 137,336 filed Apr. 4, 1980, now U.S. Pat. No. 4,284,568.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel trichothecene derivatives, to processes for their production and to their use as antitumor agents for the inhibition of malignant tumors in mammals.

2. Description of the Prior Art

The trichothecene derivatives of the present invention in general contain a 9,10 double bond and a 12,13-epoxy function. The basic skeleton and numbering system for this class of trichothecenes is shown below. ##STR1## Various examples of both naturally occurring and semisynthetic compounds of this class have been described in the literature. Illustrative of the more relevant publications are the following:

1. The compound anguidine (also called diacetoxyscirpenol) having the formula ##STR2## is disclosed as an antitumor agent in U.K. Pat. No. 1,063,255. Phase I clinical trials of anguidine in the United States have been reported in Proc. Amer. Assoc. Cancer Res. 17:90 (1976) and Proc. Amer. Assoc. Cancer Res. 18:296 (1977). Also disclosed (at least generically) are various derivatives of anguidine such as anguidol (also called scirpentriol or 3α,4β,15-trihydroxy-12,13-epoxytrichothec-9-ene), monodesacetylanguidine (presumably 15-acetoxy-3α,4β-dihydroxy--12,13-epoxytrichlothec-9-ene or monoacetoxyscirpendiol) and esters of anguidine, anguidol and monodesacetylanguidine.

Monoacetoxyscirpenol and certain esters of scirpentriol (not including any of the presently claimed compounds) are also disclosed in J. Agric. Food Chem. 24(1):97-103 (1976) as mycotoxins.

2. Japanese Published Applications J4 9,134,891 and J4 9,134,892 disclose T2 and HT2 toxins of the formula ##STR3## wherein R is --OH or ##STR4## The compounds are said to be useful as antiviral agents.

3. U.S. Pat. No. 4,129,577 discloses anguidine derivatives of the formula ##STR5## wherein R₁ is H or ##STR6## and R is an alkyl or aromatic group or is an acyl group ##STR7## in which R¹ is an aliphatic, cycloaliphatic or aromatic group or a carbamate group --CONH--R¹. The compounds are useful as cytotoxic agents.

4. U.S. Pat. No. 3,428,652 discloses anguidine derivatives of the formula ##STR8## wherein R₁ is H and R₂ is methyl or, R₁ and R₂ together represent propylene, and Hal is Cl, Br or I. The compounds are reported to have antitumor activity.

5. The 12,13-epoxytrichothecenes of the general formula ##STR9## wherein R¹ and R³ are H, OH or esterified OH and R² is OH, ═O or esterified OH are described in Biochemical and Biophysical Research Communications 57(3):838-844 (1974) as inhibitors of protein synthesis. None of the ester derivatives of the present invention are disclosed in this publication.

SUMMARY OF THE INVENTION

The present invention provides novel mono-, di- and triacylated derivatives of scirpentriol which may be represented by the general formula ##STR10## in which R₁, R₂ and R₃ are hydrogen or the residue of certain ester groups and at least one of R₁, R₂ or R₃ is other than hydrogen.

More specifically, the present invention provides (1) monoacylated ester derivatives of the formula ##STR11## in which R₃ is --COCH₂ Cl, --COCH═CHCH₃, --COCCH₃ ═CH₂, --COC₆ H₅ or --COCHClCH₃ ; ##STR12## in which R₁ is --COCH₂ Cl; and ##STR13## in which R₂ is --COCH₂ Cl; (2) diacylated ester derivatives of the formulae ##STR14## in which R₂ and R₃ are each --COCH₂ Cl; R₂ and R₃ are each --COCH═CHCH₃ ; R₂ is --COCH₂ Cl and R₃ is --COCH₃ ; or R₂ is --COCH₂ Cl and R₃ is --COCCH₃ ═CH₂ ; and ##STR15## in which R₁ and R₃ are each --COCH₂ Cl; (3) the triacylated ester derivative of the formula ##STR16## in which R₁, R₂ and R₃ are each --COCH₂ Cl; and (4) the monoacylated ester epoxy derivatives of the formula ##STR17## in which R₃ is --COCH═CHCH₃.

The ester derivatives of formula II-V are antitumor agents for treatment of malignant tumors in mammals.

As denoted by the structural formulae above, the compounds of formulae II-V all have the α-configuration at the 3-substituent and the β-configuration at the 4-substituent.

Detailed Description

The ester derivatives of the present invention may be prepared by methods known in the art. Illustrative of such methods are the reaction schemes shown below.

    ______________________________________                                         Scheme 1 - Preparation of Starting Materials                                    ##STR18##                                                                     anguidine                                                                       ##STR19##                                                                      ##STR20##                                                                      ##STR21##                                                                     Scheme 2 - Esterification of Scirpentriol                                       ##STR22##                                                                     Scheme 3 - Esterification of 3α-hydroxy-protected diol 2 to              produce diacylated esters                                                       ##STR23##                                                                      ##STR24##                                                                      ##STR25##                                                                     Scheme 4 - Esterification of starting material 5 to produce di-                acylated esters where R.sub.3 in formula IIIA is COCH.sub.3                     ##STR26##                                                                      ##STR27##                                                                      ##STR28##                                                                     Scheme 5 - Esterification of 3α-hydroxy-protected diol 2 to              produce monoacylated esters                                                     ##STR29##                                                                      ##STR30##                                                                      ##STR31##                                                                     Scheme 6 - Preparation of 4,15-diacylated esters of formula                    IIIA where R.sub.2 ≠ R.sub.3                                              ##STR32##                                                                      ##STR33##                                                                      ##STR34##                                                                      ##STR35##                                                                      ##STR36##                                                                      ##STR37##                                                                     Scheme 7 - Epoxidation to produce 9,10-epoxide                                  ##STR38##                                                                      ##STR39##                                                                     ______________________________________                                    

Explanation of Schemes 1-7

In general, the esters of the present invention may be prepared by acylation of scirpentriol or the 3-tetrahydropyran-protected derivatives 2 and 3. To prepare 3-tetrahydropyran (THP)-protected derivatives 2 and 3, the 3α-OH group of anguidine is protected as by conversion to a tetrahydropyranyl ether 1 and this 3α-OTHP derivative is then subjected to partial basic hydrolysis to give a mixture of the 4β-OH (3) and 4β,15-OH (2) derivatives.

In Scheme 2, scirpentriol may be acylated in accordance with conventional methods with a suitable acylating derivative of a carboxylic acid R-COOH to produce a mixture of the various mono-, di- and triacylated derivatives. The desired product is then separated from the product mixture as by silica gel column chromatography. The acylation is typically carried out with an acid halide or acid anhydride, preferably in the presence of an organic base such as pyridine or lutidine. An inert organic solvent such as methylene chloride may be employed or the organic base may also serve as a reaction solvent.

Scheme 3 shows the acylation of diol intermediate 2 with two or more equivalents of acylating agent followed by hydrolysis of the 3α-OTHP group to give 4,15-diacylated esters of formula IIIA having R₂ =R₃. The acylation procedure is carried out by conventional procedures such as described for Scheme 2.

Scheme 4 illustrates a procedure for preparing a 4,15-diacylated ester of formula IIIA where R₃ is --COCH₃. In this procedure starting material 5 is acylated as described above to give a 3α-THP derivative which is hydrolyzed to produce the desired product.

If diol intermediate 2 is acylated with less than two equivalents of acylating agent as in Scheme 5, there may be produced after the usual de-blocking step a mixture of 4- and 15-monoacylated products. These products can then be separated as by chromatography.

Scheme 6 shows that the monoacylated 3α-THP intermediate as produced in Scheme 5 can be treated with a second acylating agent to give after the de-protection step a diacylated ester of formula IIIA where R₂ ≠R₃.

Finally, Scheme 7 illustrates epoxidation of a 15-monoacylated ester with metachloroperbenzoic acid to give the corresponding 9,10-epoxide.

Biological Activity

Representative compounds of the present invention were tested for antitumor activity against the transplantable mouse tumors P-388 leukemia, L-1210 leukemia and Lewis lung carcinoma and the results of these tests are shown below in Tables I-XVIII. The methodology used generally followed the protocols of the National Cancer Institute (see, for example, Cancer Chemotherapy Rep. Part 3, 3:1-103 (1972)). The essential experimental details are given at the bottom of the tables.

                  TABLE 1                                                          ______________________________________                                         Effect of Compound of Example 1E on P-388 Leukemia                                                       Effect                                                                               Average                                                 Dose      MST    MST   Weight Survivors                               Compound mg/kg/day Days   % T/C Change,g                                                                              Day 5                                   ______________________________________                                         NSC-38270*                                                                              0.4       15.5   172   +0.5   6/6                                              0.2       12.5   139   +0.8   6/6                                     NSC-141537                                                                              0.8       12.5   139   +0.3   6/6                                     (Anguidine)                                                                             0.4       11.0   122   -0.3   6/6                                              0.2        9.5   100   -0.3   6/6                                              0.1        9.0   100   -0.3   6/6                                     Compound of                                                                             6.4       18.5   206   -0.3   6/6                                     Example 1E                                                                              3.2       16.0   178   -0.3   6/6                                              1.6       14.0   156   -3.1   6/6                                              0.8       12.5   139   +1.5   6/6                                              0.4       12.5   139   +0.4   6/6                                              0.2       10.5   117   0.4    6/6                                     Control  Saline     9.0   --    +0.7   10/10                                   ______________________________________                                          Tumor inoculum: 10.sup.6 ascites cells implanted i.p.                          Host: CDF.sub.1 ♂ mice.                                                   Treatment: QD 1→9.                                                      Evaluation: MST = medium survival time.                                        Effect: % T/C = MST treated/MST control × 100.                           Criteria: T/C ≧ 125 considered significant antitumor effect.            *NSC-38270 used in this and the following tables is a crude (˜40%)       preparation of olivomycin A which is used as a reference in screening of       anguidine derivatives.                                                   

                  TABLE II                                                         ______________________________________                                         Effect of Compound of Example 1C on P-388 Leukemia                                                       Effect                                                                               Average                                                 Dose      MST    MST   Weight Survivors                               Material mg/kg/day Days   % T/C Change,g                                                                              Day 5                                   ______________________________________                                         NSC-38270                                                                               0.4       14.0   156   +0.6   6/6                                              0.2       12.5   139   +0.8   6/6                                     NSC-141537                                                                              1.6       16.5   183   -0.1   6/6                                     (Anguidine)                                                                             0.8       15.0   167   +0.4   6/6                                              0.4       13.0   144   +1.3   6/6                                              0.2       11.5   128   +0.5   6/6                                              0.1       11.0   122   +1.0   6/6                                               0.05      9.5   106   +0.8   6/6                                     Compound of                                                                             6.4       14.5   161   -0.5   6/6                                     Example 1C                                                                              3.2       14.5   161   +0.3   6/6                                              1.6       12.5   139   +0.4   6/6                                              0.8       12.0   133   +0.6   6/6                                              0.4       11.0   122   +0.3   6/6                                              0.2       10.5   117   +0.3   6/6                                              0.1       10.0   111   +0.6   6/6                                               0.05     10.0   111   +0.8   6/6                                               0.025     9.0   100   +1.0   6/6                                                0.0125   9.0   100   +0.8   6/6                                     Control  0.5        9.0   --    +0.5   10/10                                   ______________________________________                                          Tumor inoculum: 10.sup.6 ascites cells implanted i.p.                          Host: CDF.sub.1 ♀ mice.                                                 Treatment: QD 1→9.                                                      Evaluation: MST = medium survival time.                                        Effect: % T/C = MST treated/MST control × 100.                           Criteria: T/C ≧ 125 considered significant antitumor effect.      

                  TABLE III                                                        ______________________________________                                         Effect of Compound of Example 9 on P-388 Leukemia                                                        Effect                                                                               Average                                                 Dose      MST    MST   Weight Survivors                               Compound mg/kg/day Days   % T/C Change,g                                                                              Day 5                                   ______________________________________                                         NSC-38270                                                                               0.4       13.0   144   -0.7   6/6                                              0.2       11.0   122   -0.5   6/6                                     NSC-141537                                                                              3.2       17.5   194   +0.6   6/6                                     (Anguidine)                                                                             1.6       17.5   194   +0.2   6/6                                              0.8       15.0   167   +0.4   6/6                                              0.4       14.5   161   +0.5   6/6                                              0.2       13.5   150   +1.0   6/6                                              0.1       11.0   122   +0.1   6/6                                               0.05     11.0   122   +0.2   5/5                                               0.025     9.5   106   +0.5   6/6                                     Compound of                                                                             3.2       16.5   183   +0.8   6/6                                     Example 9                                                                               1.6       16.0   178   +1.0   6/6                                              0.8       16.0   178   +1.3   6/6                                              0.4       14.5   161   +0.4   6/6                                              0.2       12.0   133   +0.7   6/6                                              0.1       12.0   133   +0.8   6/6                                               0.05     10.5   117   +0.3   6/6                                               0.025    10.5   117   +0.3   6/6                                                0.0125  10.0   111   +0.3   6/6                                                0.00625 10.0   111   +0.8   6/6                                     Control  DMSO-      9.0   --    +0.4   10/10                                            HPC                                                                   ______________________________________                                          Tumor inoculum: 10.sup.6 ascites cells implanted i.p.                          Host: CDF.sub.1 ♀ mice.                                                 Treatment: QD 1→9.                                                      Evaluation: MST = medium survival time.                                        Effect: % T/C = MST treated/MST control × 100.                           Criteria: T/C ≧ 125 considered significant antitumor effect.      

                  TABLE IV                                                         ______________________________________                                         Effect of Derivatives on P-388 Leukemia                                                                  Effect                                                                               Average                                                 Dose      MST    MST   Weight Survivors                               Compound mg/kg/day Days   % T/C Change,g                                                                              Day 5                                   ______________________________________                                         NSC-38270                                                                               0.4       12.5   139   +0.3   6/6                                              0.2       11.0   122   +1.3   6/6                                     NSC-141537                                                                              1.6       17.0   189   +0.8   6/6                                     (anguidine)                                                                             0.8       15.0   167   +1.0   6/6                                              0.4       13.5   150   +1.6   6/6                                              0.2       13.0   144   +1.5   6/6                                              0.1       11.0   122   +1.4   6/6                                               0.05     11.0   122   +1.8   6/6                                     Compound of                                                                             6.4       14.0   156   +0.9   6/6                                     Example 1D                                                                              3.2       13.0   144   +1.3   6/6                                              1.6       12.5   139   +2.8   6/6                                              0.8       12.5   139   +2.3   6/6                                              0.4       11.5   128   +1.6   6/6                                              0.2       10.5   117   +1.2   6/6                                     Compound of                                                                             6.4       17.5   194   +1.2   6/6                                     Example 1B                                                                              3.2       14.0   156   +3.0   5/5                                              1.6       13.0   144   +1.5   6/6                                              0.8       12.0   133   +1.7   6/6                                              0.4       12.0   133   +1.7   6/6                                              0.2       10.0   111   +1.4   6/6                                     Compound of                                                                             6.4       16.0   178   -0.9   6/6                                     Example 8                                                                               3.2       14.0   156   - 0.6  6/6                                              1.6       13.0   144   -0.1   6/6                                              0.8       12.0   133   -0.3   6/6                                              0.4       12.5   139   -0.7   6/6                                              0.2       11.5   128   -0.3   6/6                                              0.1       13.0   144   -0.6   6/6                                              0.5       11.0   122   -0.5   6/6                                               0.025    10.0   111   -0.1   6/6                                                0.0125  10.0   111   -0.4   6/6                                     Control  Saline           --    +0.5   10/10                                   ______________________________________                                          Tumor inoculum: 10.sup.6 ascites cells implanted i.p.                          Host: CDF.sub.1 ♂ mice.                                                   Treatment: QD 1→9.                                                      Evaluation: MST = medium survival time.                                        Effect: % T/C = MST treated/MST control × 100.                           Criteria: T/C ≧ 125 considered significant antitumor effect.      

                  TABLE V                                                          ______________________________________                                         Effect of Compound of Example 1A on P-388 Leukemia                                                       Effect                                                                               Average                                                 Dose      MST    MST   Weight Survivors                               Compound mg/kg/day Days   % T/C Change,g                                                                              Day 5                                   ______________________________________                                         NSC-38270                                                                               0.4       13.0   144   +0.1   5/5                                              0.2       11.0   122   +0.3   6/6                                     NSC-141537                                                                              3.2       17.5   194   +0.7   6/6                                     (anguidine)                                                                             1.6       17.0   189   +0.7   6/6                                              0.8       14.0   156   +1.8   6/6                                              0.4       13.5   150   +1.9   6/6                                              0.2       12.0   133   +0.8   6/6                                              0.1       11.0   122   +1.0   6/6                                               0.05     11.5   128   +1.2   6/6                                               0.025    10.0   111   +1.3   6/6                                     Compound of                                                                             12.8      Tox    Tox   Tox    2/6                                     Example 1A                                                                              6.4       Tox    Tox   Tox    2/6                                              3.2       17.5   194   +0.5   6/6                                              1.6       17.0   189   +1.3   6/6                                              0.8       14.5   161   +1.8   6/6                                              0.4       16.0   178   +1.4   6/6                                              0.2       14.0   156   +0.8   6/6                                              0.1       13.0   144   +1.3   6/6                                               0.05     13.5   150   +1.3   6/6                                               0.025    12.5   139   +1.4   6/6                                                0.0125  10.5   117   +1.3   6/6                                                0.00625 10.5   117   +1.7   6/6                                     Control  Saline     9.0   --    +0.6   10/10                                   ______________________________________                                          Tumor inoculum: 10.sup.6 ascites cells implanted i.p.                          Host: CDF.sub.1 ♀ mice.                                                 Treatment: Daily, QD 1→9.                                               Tox: Toxicity <4/6 survivors, Day 5.                                           Evaluation: MST = median survival time.                                        Effect: % T/C = MST control × 100.                                       Criteria: T/C ≧ 125 considered significant antitumor effect.      

                  TABLE VI                                                         ______________________________________                                         Effect of Compound of Example 2 on P-388 Leukemia                                                        Effect                                                                               Average                                                 Dose      MST    MST   Weight Survivors                               Material mg/kg/day Days   % T/C Change,g                                                                              Day 5                                   ______________________________________                                         NSC-38270                                                                               0.4       13.0   153   +0.4   6/6                                     A649     0.2       10.0   118   +1.2   6/6                                     NSC-141537                                                                              1.6       16.0   188   +1.0   6/6                                     Anguidine                                                                               0.8       14.0   165   +0.9   6/6                                              0.4       13.0   153   +1.3   6/6                                              0.2       12.0   141   +1.0   6/6                                              0.1       11.0   129   +0.4   6/6                                              0.05      10.0   118   +1.1   6/6                                     Compound of                                                                             6.4       18.0   212   -0.3   5/6                                     Example 2                                                                               3.2       16.5   194   +1.0   6/6                                              1.6       15.5   182   +1.1   6/6                                              0.8       14.0   165   +1.3   6/6                                              0.4       13.0   153   +0.8   6/6                                              0.2       13.0   153   +0.2   6/6                                              0.1       11.5   135   +0.8   6/6                                              0.05      12.5   147   +0.9   6/6                                              0.025     10.0   118   +1.1   6/6                                              0.0125     9.0   106   +2.4   6/6                                              0.00625    9.0   106   +2.3   6/6                                              0.003125   9.0   106   +3.1   6/6                                     Control  Saline     8.5   --    +3.1   10/10                                   ______________________________________                                          Tumor inoculum: 10.sup.6 ascites cells implanted i.p.                          Host: CDF.sub.1 ♀ mice.                                                 Treatment: QD 1→9.                                                      Evaluation: MST = median survival time.                                        Effect: % T/C = MST treated/MST control × 100.                           Criteria: T/C ≧ 125 considered significant antitumor effect.      

                  TABLE VII                                                        ______________________________________                                         Effect of Compound of Example 2 on P-388 Leukemia                                                Dose,        Ef-                                                               IP           fect Average                                                                               Sur-                                                  mg/          MST  Weight vivors                                                kg/     MST  %    Change,                                                                               Day 5                               Material                                                                               Treatment day     Days I/C  g      (30)                                ______________________________________                                         NSC 38270                                                                              Days 1 → 9                                                                        0.4     11.5 128  -1.5   6/6                                                   0.2     10.0 111  -0.9   6/6                                 Anguidine                                                                              Day 1 only                                                                               20      Tox  Tox  Tox    0/6                                 NSC               16      Tox  Tox  Tox    0/6                                 141537            12      Tox  Tox  Tox    2/6                                                    8      Tox  Tox  Tox    3/6                                         Days 1 → 5                                                                         5      Tox  Tox  Tox    3/6                                                    4      15.0 167  +0.3   6/6                                                    3      14.0 156  +0.3   6/6                                                    2      13.0 144  +0.2   6/6                                         Days 1 → 9                                                                        2.4     16.0 178  +1.3   6/6                                                   1.6     16.0 178  +0.6   5/5                                 Compound                                                                               Day 1 only                                                                               60      Tox  Tox  Tox    0/6                                 of Example        45      Tox  Tox  Tox    0/6                                 2                 30      Tox  Tox  Tox    1/6                                                   20      Tox  Tox  Tox    0/6                                         Days 1 → 5                                                                        12      Tox  Tox  Tox    2/6                                                   10      Tox  Tox  Tox    1/6                                                    8      13.0 144  -1.5   5/6                                                   6.4     13.5 150  -0.8   4/6                                         Days 1 → 9                                                                        9.0     Tox  Tox  Tox    3/6                                                   6.4     12.0 133  +0.3   6/6                                 Control           Saline   9.0 --   +2.2   10/10                               ______________________________________                                          Tumor inoculum: 10.sup.6 ascites cells implanted i.p.                          Host: CDF.sub.1 ♀ mice.                                                 Tox: <4/6 survivors Day 5.                                                     Evaluation: MST = median survival time.                                        Effect: % T/C = (MST treated/MST control) × 100.                         Criteria: % T/C ≧ 125 considered significant antitumor activity.  

                  TABLE VIII                                                       ______________________________________                                         Effect of Compound of Example 2 on P-388 Leukemia                                                        Effect                                                                               Average Sur-                                            Dose      MST    MST   Weight  vivors                                 Material mg/kg/inj Days   % T/C Change, g                                                                              Day 5                                  ______________________________________                                         NSC-141537                                                                              1.6       11.0   183   +0.5    6/6                                    (Anguidine)                                                                             0.8       8.5    142   +1.2    6/6                                             0.4       8.5    142   +1.2    6/6                                             0.2       8.0    133   +1.5    6/6                                             0.1       7.0    117   +1.9    6/6                                             0.05      7.0    117   +2.6    6/6                                    Compound of                                                                             12.8      8.5    142   -0.9    4/6                                    Example 2                                                                               6.4       10.5   175   -0.2    6/6                                             3.2       9.5    158   +1.0    6/6                                             1.6       9.5    158   +1.8    6/6                                             0.8       8.5    142   -1.3    6/6                                             0.4       8.5    142   -0.7    6/6                                             0.2       7.0    117   +0.3    6/6                                             0.1       7.0    117   +0.3    6/6                                    Control  Saline    6.0    --    +2.5    10/10                                  ______________________________________                                          Tumor inoculum: 10.sup.6 ascites cells implanted i.p.                          Host: BDF.sub.1 ♀ mice.                                                 Treatment: Daily, QD 1 → 9.                                             Evaluation: MST = median survival time.                                        Effect: % T/C =  MST treated/MST control × 100.                          Criteria: T/C ≧ 125 considered significant antitumor effect.      

                  TABLE IX                                                         ______________________________________                                         Effect of Compound of Example 4 on P-388 Leukemia                                                        Effect                                                                               Average Sur-                                            Dose      MST    MST   Weight  vivors                                 Material mg/kg/day Days   % T/C Change,g                                                                               Day 55                                 ______________________________________                                         NSC-38270                                                                               0.4       12.0   133   +0.7    6/66                                   (A-649)  0.2       11.0   122   -0.4    6/66                                   NSC-141537                                                                              1.6       17.0   189   +1.3    5/55                                   (anguidine)                                                                             0.8       14.0   156   +1.7    5/66                                            0.4       14.0   156   +1.9    6/66                                            0.2       12.0   133   +1.5    6/66                                            0.1       11.5   128   +0.8    6/66                                            0.05      10.0   111   +0.3    6/66                                   Compound of                                                                             6.4       Tox    Tox   Tox     1/66                                   Example 4                                                                               3.2       20.0   222   -1.9    5/66                                            1.6       17.0   189   -0.9    6/66                                            0.8       14.5   161   +0.9    6/66                                            0.4       13.0   144   +0.2    5/66                                            0.2       12.5   139   0       6/66                                            0.1       13.0   144   0       6/66                                            0.05      11.0   122   0.8     6/66                                            0.025     11.0   122   -0.3    6/66                                            0.0125    11.0   122   -0.4    6/66                                   Control  Saline     9.0   --    +0.3    10/730                                 ______________________________________                                          Tumor inoculum: 10.sup.6 ascites cells implanted i.p.                          Host: CDF.sub.1 ♀ mice.                                                  Treatment: Daily, QD 1 9.                                                     Tox: Toxicity, 4/6 survivors Day 5.                                            Evaluation: MST = median survival time.                                        Effect: % T/C = MST treated/MST control × 100.                           Criteria: T/C 125 considered significant antitumor effect                

                  TABLE X                                                          ______________________________________                                         Effect of Compound of Example 4 on L-1210 Leukemia                                    Dose,                 Average                                                  IP            Effect  Weight                                                   mg/kg/ MST    MST     Change Survivors                                  Material day      Days   % T/C g      Day 5 (30)                               ______________________________________                                         Anguidine                                                                               2.0      11.0   157   -0.8   6/6                                      NSC 141537                                                                              1.6      11.0   157   -0.3   6/6   (1/6)                                       1.2      11.0   157   -0.3   6/6                                               0.8      11.0   157   -0.3   6/6                                               0.4      10.0   143   -0.1   6/6                                               0.2       9.0   129   +0.5   6/6   (1/6)                              Compound of                                                                             1.6      12.0   171   -0.8   4/6   (2/6)                              Example 4                                                                               0.8      10.0   143   -0.3   6/6                                               0.4       9.5   136   -0.8   6/6                                               0.2       9.0   129   -0.3   6/6                                      Control  Saline    7.0   --    +0.9   10/10                                    ______________________________________                                          Tumor inoculum: 10.sup.6 ascites cells implanted ip                            Host: BDF.sub.1 ♀ mice.                                                 Treatment: QD1 → 9                                                      Tox: <4/6 mice alive on Day 5                                                  Evaluation: MST = median survival time.                                        Effect: % T/C = (MST treated/MST control) × 100.                         Criteria: % T/C ≧ 125 considered significant antitumor activity.  

                  TABLE XI                                                         ______________________________________                                         Effect of Derivatives on L-1210 Leukemia                                                                 Effect                                                                               Average Sur-                                            Dose      MST    MST   Weight  vivors                                 Material mg/kg/day Days   % T/C Change, g                                                                              Day 5                                  ______________________________________                                         Anguidine                                                                               2.0       6.0     86   -0.9    4/6                                    NSC 141537                                                                              1.6       6.0     86   -1.3    6/6                                             1.2       11.0   157   -1.1    5/6                                             0.8       11.0   157   +1.0    5/6                                    Compound of                                                                             4.0       Tox    Tox   Tox     0/6                                    Example 4                                                                               3.2       Tox    Tox   Tox     0/6                                             2.4       Tox    Tox   Tox     2/6                                             1.6       Tox    Tox   Tox     3.6                                    Compound of                                                                             2.4       6.0     86   -1.5    5/6                                    Example 3                                                                               1.6       6.0     86   -1.3    6/6                                             1.2       8.0    114   -0.6    6/6                                             0.8       12.0   171   -2.6    6/6                                    Compound of                                                                             0.6       Tox    Tox   Tox     3.6                                    Example 6                                                                               0.4       10.0   143   -1.1    5/6                                             0.3       10.5   150   -1.0    4/6                                             0.2       10.0   143   +0.1    5/6                                    Control  Saline    7.0    --    +2.4    10/10                                  ______________________________________                                          Tumor inoculum: 10.sup.6 ascitic cells implanted ip                            Host: BDF.sub.1 ♀ mice.                                                 Treatment: QD 1 → 9                                                     Tox: <4/6 survivors Day 5                                                      Evaluation: % T/C = MST treated/MST control × 100.                       Criteria: % T/C ≧ 125 considered significant antitumor effect.    

                  TABLE XII                                                        ______________________________________                                         Effect of Compound of Example 3 on P-388 Leukemia                                                        Effect                                                                               Average Sur-                                            Dose      MST    MST   Weight  vivors                                 Material mg/kg/day Days   % T/C Change, g                                                                              Day 5                                  ______________________________________                                         NSC-38270                                                                               0.4       12.5   139   -0.6    6/6                                             0.2       11.0   122   +0.3    6/6                                    NSC-141537                                                                              1.6       15.0   167   -0.2    6/6                                    (anguidine)                                                                             0.8       14.0   156   +0.5    6/6                                             0.4       17.0   189   +0.2    6/6                                             0.2       16.5   183   -0.5    6/6                                             0.1       11.0   122   +0.7    6/6                                             0.05      10.5   117   +0.6    6/6                                    Compound of                                                                             6.4       7.0     78   -2.0    5/6                                    Example 3                                                                               3.2       7.5     83   -1.0    6/6                                             1.6       20.0   222   -0.6    6/6                                             0.8       19.5   217   +0.1    6/6                                             0.4       17.0   189   +0.5    /66                                             0.2       15.5   172   -0.1    6/6                                             0.1       14.5   161   +0.1    6/6                                             0.05      13.0   144   +0.1    6/6                                             0.025     12.0   133   -0.2    6/6                                             0.0125    10.5   117   +0.6    6/6                                    Control  Saline    9.0    --    0       10/10                                  ______________________________________                                          Tumor inoculum: 10.sup.6 ascites cells implanted i.p.                          Host: CDF.sub.1 ♀ mice.                                                 Treatment: QD 1 → 9.                                                    Tox: Toxicity, <4/6 survivors, Day 5.                                          Evaluation: MST = median survival time.                                        Effect: % T/C = MST treated/MST control × 100.                           Criteria: T/C ≧ 125 considered significant antitumor effect.      

                  TABLE XIII                                                       ______________________________________                                         Effect of Compound of Example 5 on P-388 Leukemia                                                        Effect                                                                               Average Sur-                                            Dose      MST    MST   Weight  vivors                                 Material mg/kg/day Days   % T/C Change, g                                                                              Day 5                                  ______________________________________                                         NSC-38270                                                                               0.4       11.0   122   -0.4    6/6                                             0.2       9.5    106   +0.1    6/6                                    Compound of                                                                             6.4       15.0   167   -0.4    5/6                                    Example 5                                                                               3.2       14.5   161   0       6/6                                             1.6       13.0   144   +1.5    5/6                                             0.8       10.0   111   +0.8    6/6                                             0.4       9.0    100   +1.2    6/6                                             0.2       9.5    106   +2.8    6/6                                             0.1       10.0   111   +2.9    6/6                                              0.05     9.0    100   +3.1    6/6                                    NSC-141537                                                                              1.6       15.0   167   +1.5    6/6                                    (Anguidine)                                                                             0.8       15.0   167   +1.6    6/6                                             0.4       14.0   156   +1.2    6/6                                             0.2       12.0   133   +0.8    6/6                                             0.1       10.5   117   +1.4    6/6                                              0.05     10.0   111   +1.5    6/6                                    Control  Saline    9.0    --    +3.7    10/10                                  ______________________________________                                          Tumor inoculum: 10.sup.6 ascites cells implanted i.p.                          Host: CDF.sub.1 ♀ mice.                                                 Treatment: QD 1 → 9.                                                    Evaluation: MST = median survival time.                                        Effect: % T/C = MST treated/MST control × 100.                           Criteria: T/C ≧ 125 considered significant antitumor effect.      

                  TABLE XIV                                                        ______________________________________                                         Effect of Compound of Example 6 on P-388 Leukemia                                                        Effect                                                                               Average Sur-                                            Dose      MST    MST   Weight  vivors                                 Material mg/kg/day Days   % T/C Change, g                                                                              Day 5                                  ______________________________________                                         NSC-38270                                                                               0.4       11.0   138   -1.0    6/6                                             0.2       10.0   125   -0.2    6/6                                    Anguidine                                                                               1.6       15.0   188   +0.6    6/6                                    NSC 141537                                                                              0.8       13.0   163   +0.7    6/6                                             0.4       13.0   163   +0.3    6/6                                             0.2       12.0   150   +0.8    6/6                                             0.1       10.0   125   +0.1    6/6                                              0.05     10.0   125   +0.4    6/6                                    Compound of                                                                             12.8      Tox    Tox   Tox     0/6                                    Example 6                                                                               6.4       Tox    Tox   Tox     0/6                                             3.2       Tox    Tox   Tox     0/6                                             1.6       Tox    Tox   Tox     0/6                                             0.8        6.0    75   -1.9    4/6                                             0.4       18.0   225   -1.1    6/6                                             0.2       15.5   194   -0.5    6/6                                             0.1       14.0   175   -0.7    6/6                                    Control  Saline     8.0   --    -0.4    10/10                                  ______________________________________                                          Tumor inoculum: 10.sup.6 ascitic cells implanted i.p.                          Host: CDF.sub.1 ♀ mice.                                                 Treatment: Daily, QD 1 → 9.                                              Tox: <4/6 survivors Day 5.                                                    Evaluation: MST = median survival time.                                        Effect: % T/C = (MST treated/MST control) × 100.                         Criteria: % T/C ≧ 125 considered significant antitumor activity.  

                  TABLE XV                                                         ______________________________________                                         Effect of Compound of Example 10 on P-388 Leukemia                                                       Effect                                                                               Average Sur-                                            Dose      MST    MST   Weight  vivors                                 Material mg/kg/day Days   % T/C Change, g                                                                              Day 5                                  ______________________________________                                         NSC 38270                                                                               0.4       10.5   117   -1.2    6/6                                             0.2       10.5   117   -0.8    6/6                                    Anguidine                                                                               1.6       17.5   195   -0.7    6/6                                    NSC 141537                                                                              0.8       15.0   167   +0.7    6/6                                             0.4       14.0   156   +0.2    6/6                                             0.2       12.0   133   -0.3    6/6                                             0.1       10.5   117   +0.8    6/6                                              0.05     10.5   117   +0.4    6/6                                    Compound of                                                                             12.8      16.5   183   -0.8    6/6                                    Example 10                                                                              6.4       15.0   167   +0.3    6/6                                             3.2       16.0   178   +0.8    6/6                                             1.6       12.0   133   -0.1    6/6                                             0.8       12.0   133   +0.2    6/6                                             0.4       11.0   122   +0.7    6/6                                    Control  Saline     9.0   --    -1.8    10/10                                  ______________________________________                                          Tumor inoculum: 10.sup.6 ascitic cells implanted ip                            Host: CDF.sub.1 ♂ mice.                                                   Treatment: QD 1 → 9.                                                    Tox: < 4/6 survivors Day 5                                                     Evaluation: MST = median survival time                                         Effect: % T/C = (MST treated/MST control) × 100.                         Criteria: % T/C ≧ 125 considered significant antitumor activity.  

                  TABLE XVI                                                        ______________________________________                                         Effect of Compound of Example 7 on P-388 Leukemia                                                                      Sur-                                                             Effect                                                                               Average vivors                                          Dose, IP  MST    MST   Weight  Day 5                                  Material mg/kg/inj Days   % T/C Change, %                                                                              (30)                                   ______________________________________                                         NSC 38270                                                                               0.4       9.0    100   +0.3    6/6                                             0.2       9.0    100   +2.8    6/6                                    Anguidine                                                                               1.6       14.5   161   +1.4    6/6                                    NSC 141537                                                                              0.8       13.0   144   +1.7    6/6                                             0.4       12.0   133   +1.8    6/6                                             0.2       10.0   111   +1.4    6/6                                             0.1       9.0    100   +2.3    6/6                                              0.05     9.0    100   +2.6    6/6                                    Compound of                                                                             12.8      Tox    Tox   Tox     0/6                                    Example 7                                                                               6.4       Tox    Tox   Tox     0/6                                             3.2       Tox    Tox   Tox     0/6                                             1.6       18.0   200   -1.3    4/6                                             0.8       17.5   194   -0.5    6/6                                             0.4       15.0   167   -0.1    6/6                                             0.2       12.0   133   +0.3    6/6                                             0.1       12.0   133   +0.3    6/6                                              0.05     11.0   122   +1.2    6/6                                              0.025    10.0   111   +1.1    5/6                                    Control  Saline    9.0    --    +4.0    10/10                                  ______________________________________                                          Tumor inoculum: 10.sup.6 ascites cells implanted i.p.                          Host: CDF.sub.1 ♀ mice.                                                 Treatment: QD1 → 9                                                      Evaluation: MST = median survival time.                                        Effect: % T/C + (MST treated/MST Control) × 100.                         Criteria: % T/C 125 considered significant antitumor effect.             

                  TABLE XVII                                                       ______________________________________                                         Effect of Compound of Example 7 on L1210 Leukemia                                                                      Sur-                                                             Effect                                                                               Average vivors                                          Dose,IP   MST    MST   Weight  Day 5                                  Material mg/kg/day Days   % T/C Change, g                                                                              (30)                                   ______________________________________                                         Anguidine                                                                               2.4       12.0   200   +1.0    6/6                                    NSC 141537                                                                              2.0       11.0   183   +1.9    6/6                                             1.6       10.0   167   +1.4    6/6                                             1.2       10.0   167   +0.9    6/6                                             0.8       10.0   167   +0.9    6/6                                             0.4       9.0    150   +0.1    6/6                                             0.2       8.0    133   +1.3    6/6                                             0.1       8.0    133   +0.8    6/6                                    Compound of                                                                             2.4       TOX    TOX   TOX     1/6                                    Example 7                                                                               2.0       7.0    117   -1.2    3/6                                             1.6       10.0   167   -1.7    5/6                                             1.2       9.5    158   -1.1    6/6                                             0.8       10.0   167   -0.5    6/6                                             0.4       10.0   167   -0.8    6/6                                             0.2       9.0    150   -0.5    5/6                                             0.1       8.0    133   +0.5    6/6                                    Control  Saline    6.0    --    +2.6    10/10                                  ______________________________________                                          Tumor inoculum: 10.sup.6 ascites cells implanted, ip.                          Host: BDF.sub.1 ♀ mice.                                                 Treatment: QD 1 → 9.                                                    Evaluation: MST = median survival time.                                        Effect: % T/C = (MST treated/MST control). × 100                         Criteria: % T/C ≧ 125 considered significant antitumor activity.  

                  TABLE XVIII                                                      ______________________________________                                         Effect of Compound of Example 7 on Lewis Lung Carcinoma                                                  Effect                                                                               Average                                                 Dose, IP  MST    MST   Weight  Survivors                              Material mg/kg/day Days   % T/C Change  Day 5 (60)                             ______________________________________                                         Anguidine                                                                               1.6       21.0   124   +2.2    10/10                                  NSC 141537                                                                              0.8       21.0   124   +1.8    10/10                                           0.4       23.0   135   +1.4    10/10                                  Compound of                                                                             2.0       21.5   126   -0.5     6/10                                  Example 7                                                                               1.5       21.5   126   -0.3    10/10                                           1.0       22.0   129   +0.5    10/10                                           0.5       22.0   129   +1.2    10/10                                  Control  Saline    17.0   --    -0.6    10/10                                  ______________________________________                                          Tumor inoculum: 10.sup.6 tumor brei cells, ip.                                 Host: BDF.sub.1 ♂ mice.                                                   Treatment: QD 1 → 9. Tox: <6/10 mice alive on Day 5.                    Evaluation: MST = median survival time.                                        Effect: % T/C = (MST treated/MST control) × 100.                         Criteria: % T/C ≧ 125 considered significant antitumor activity.  

Explanation

Each of the 14 derivatives of the present invention was evaluated in mice against P388 leukemia (ip) in parallel with anguidine itself, using a qd 1→9 dosing schedule (ip). The compounds were all found to be active and comparable to anguidine with respect to this tumor system.

The compound of Example 2 was evaluated twice against P388 leukemia (Tables VI and VII). As can be seen, the compound for some as yet unexplained reason appeared significantly more active in one test than the other.

Five compounds were evaluated in mice against L1210 leukemia (ip). All of them were active with maximum T/C values of between 150% and 175% using a qd 1→9 dosing schedule.

The compound of Example 7 was also evaluated in mice against Lewis lung carcinoma (ip). It produced a maximum T/C of 129% when given qd 1→9.

The following examples are not limiting but are intended to be illustrative of this invention. SKELLYSOLVE B is a commercially available petroleum solvent (Skelly Oil Co.) comprising isomeric hexanes and having a boiling point of 60°-68° C. The main component of SKELLYSOLVE B is n-hexane. Unless otherwise indicated, all melting points below are uncorrected, all temperatures are in degrees Celsius and all solvent percentages are by volume. The silica gel used in the examples (unless otherwise indicated) is SILICAR CC-7 (trademark of Mallinckrodt Chemical Works).

PREPARATION OF STARTING MATERIALS Preparation 1 4β,15-Diacetoxy-3α-O-(2'-tetrahydropyranyl)-12,13-epoxytrichothec-9-ene

A mixture of 4β,15-diacetoxy-3α-hydroxy-12,13-epoxytrichothec-9-ene (12.81 g, 35 mmol), 2,3-dihydro-4H-pyran (17.5 ml, 189 mmol), and p-toluenesulfonic acid (70 mg, 0.35 mmol) in 150 ml of CH₂ Cl₂ was stirred at room temperature for 2 h. After addition of 2.1 g of K₂ CO₃, the reaction mixture was diluted with 400 ml of CH₂ Cl₂ and washed with saturated NaHCO₃ solution and brine. Drying over K₂ CO₃ and removal of the solvent gave a colorless oil which crystallized slowly from petroleum ether to give 11.30 g (72%) of solid. m.p. 93°-94° C.; IR(KBr): 2976, 1746, 1249, 1080, 1040, 988 cm⁻¹.

Anal. Calc'd for C₂₄ H₃₄ O₈ : C, 63.98; H, 7.61. Found: C, 64.35; H, 7.58.

Preparation 2 3α,4β,15-Trihydroxy-12,13-epoxytrichothec-9-ene

4β,15-Diacetoxy-3α-hydroxy-12,13-epoxytrichothec-9-ene (15 g) was stirred for 20 minutes in 300 ml of methanol with 900 ml of 0.3 N sodium hydroxide (Sigg et al. Helv. Chim. Acta, 48, 962-988 (1965). The solution was placed on a column containing 1 kg of DOWEX 50 (H⁺ cycle) prepared with 20% methanol in water. The column was eluted with 3 l of the same solvent, the eluate concentrated, and the residual aqueous solution freeze-dried. The powder was dissolved in methanol, mixed with 10 g of silica gel, and dried in vacuo. The dry silica gel mixture was placed on a column of fresh silica gel (2.5×100 cm) and eluted with methylene chloride with increasing amounts of methanol. Fractions appearing homogeneous on TLC plates were dried and crystallized from ethyl acetate. Yield: 7.3 g, m.p. 194°-195° C. IR(KBr): 3490, 3450, 3390, 2990-2900 (four peaks), 1675, 960 and 950 cm⁻¹. [α]_(D) ²² =-15.4° (c=1, acetone).

Anal. Calc'd for C₁₅ H₂₂ O₆ : C, 63.81; H, 7.86. Found: C, 63.71; H, 7.80.

Alternatively, the 3-O-tetrahydropyranyl derivative (Preparation 3 below) (1 g) was stirred for four hours in 115 ml of 95% ethanol and 23 ml of 1 N HCl. The solution was azeotropically distilled with the addition of absolute ethanol, the concentrated ethanolic solution diluted with diethyl ether, and the resulting title product separated from ethyl acetate as a gum.

Preparation 3 4β,15-Dihydroxy-3α-O-(2'-tetrahydropyranyl)-12,13-epoxytrichothec-9-ene

To a solution of 4β,15-diacetoxy-3-O-(2'-tetrahydropyranyl)-12,13-epoxytrichothec-9-ene (1.067 g, 2.37 mmol) in 40 ml of tetrahydrofuran and methanol (5:3 v/v) was added 20 ml of 0.3 N NaOH solution. After 2.5 h of stirring at room temperature, an additional 20 ml of 0.3 N NaOH solution was introduced, and stirring was continued for 18.5 h. The resulting solution was diluted with CH₂ Cl₂ (200 ml) and washed with water. The aqueous layer was reextracted with CH₂ Cl₂ (2×50 ml). The combined CH₂ Cl₂ layers were washed with brine and dried over K₂ CO₃. Removal of the solvent gave 891 mg of foam, which was subsequently chromatographed on silica gel. Elution with 1% methanol-CH₂ Cl₂ gave 46 mg (5%) of 15-acetoxy-4β-hydroxy-3α-O-(2'-tetrahydropyranyl)-12,13-epoxytrichothec-9-ene. A further elution with 5 % methanol-CH₂ Cl₂ gave 808 mg (93%) of the title compound as an amorphous solid. IR(KBr): 3457, 2943, 1445, 1135, 1125, 1078, 1035, 1020, 978, 957 cm⁻¹.

Preparation 4 15-Acetoxy-4β-hydroxy-3α-O-(2'-tetrahydropyranyl)-12,13-epoxytrichothec-9-ene

To a solution of 4β,15-diacetoxy-3α-O-(2'-tetrahydropyranyl)-12,13-epoxytrichothec-9-ene (31.14 g, 69.2 mmol) in 800 ml of methanol and tetrahydrofuran (1:1 v/v) was added 400 ml of 1.31 N NH₄ OH solution. After 3 days stirring at room temperature, 10 ml of concentrated NH₄ OH solution was added to the reaction mixture. Stirring was continued for an additional 4 days. The volume of the resulting solution was reduced to 500 ml. Extraction with CH₂ Cl₂, washing with brine, and removal of the solvent gave 37 g of a slightly yellow oil. Chromatography on silica gel (elution with 1% methanol-CH₂ Cl₂) gave 10.65 g (38%) of the title compound as an oil. The NMR and IR spectra of this material were consistent with the structure of the title compound. IR(KBr): 3430, 2970, 2950, 2875, 1744, 1720, 1270, 1248, 1126, 1080, 1040, 972 cm⁻¹.

EXAMPLE 1 A. 15-Chloroacetoxy-3α,4β-dihydroxy-12,13-epoxytrichothec-9-ene

Scirpentriol (3α,4β,15-trihydroxy-12,13-epoxytrichothec-9-ene) (7 g) was dissolved in 30 ml. of 2,6-lutidine and treated with 10.75 g of chloroacetic anhydride. After 18 hours the solution was poured on ice. The product of the usual isolation procedure was chromatographed on a column of silica gel (2×75 cm) by gradient elution with methylene chloride/methanol. The emerging (Rf 0.1, 0.35, 0.58, 0.80, 0.90) fractions were analyzed on TLC plates appropriately combined, dried and crystallized from ethyl acetate-ether-hexane. Fractions, Rf 0.35 gave 0.640 mg of title product. M.p. 173°. IR(KBr): 3520, 3380, 3800-2900 (4 peaks) 1725, 1295, 1060.

Anal. Calcd for C₁₇ H₂₃ O₆ Cl: C, 56.91; H, 6.46; Cl, 9.88. Found: C, 56.85; H, 6.39; Cl, 9.65.

B. 3α-Chloroacetoxy-4β,15-dihydroxy-12,13-epoxytrichothec-9-ene

Fractions Rf 0.58 from Example 1, Part A, gave 480 mg. of title product; m.p. 170°. IR(KBr): 3500, 3000-2900 (five peaks) 1758, 1745, 1210, 1170, 1055, 918 cm⁻¹.

Anal. Calcd for C₁₇ H₂₃ O₆ Cl: C, 56.91; H, 6.46; Cl, 9.88. Found: C, 57.03; H, 6.46; Cl, 9.85.

C. 3α,15-Bis(chloroacetoxy)-4β-hydroxy-12,13-epoxytrichothec-9-ene

Fractions Rf 0.8 from Example 1, Part A, gave 1.5 g title product; m.p. 161°. IR(KBr): 3480, 3060-2840 (seven peaks) 1765, 1735, 1295, 1200, 1165 cm⁻¹.

Anal. Calcd for C₁₉ H₂₄ O₇ Cl₂ : C, 52.18; H, 5.99; Cl, 16.21. Found: C, 52.41; H, 5.55; Cl, 16.44.

D. 4β-Chloroacetoxy-3α,15-dihydroxy-12,13-epoxytrichothec-9-ene

Fractions with Rf 0.68 from Example 1, Part A, gave 205 mg. of title product. The structure of the product was confirmed by PMR spectroscopy.

E. 3α,4β,15-Tri(chloroacetoxy)-12,13-epoxytrichothec-9-ene

Fractions with Rf 0.9 from Example 1, Part A, gave 95 mg. of title product. The structure of the product was confirmed by PMR spectroscopy.

EXAMPLE 2 4β,15-Bis(chloroacetoxy)-3α-hydroxy-12,13-epoxytrichothec-9-ene A. 4β,15-Bis(chloroacetoxy)-3α-0-(2'-tetrahydropyranyl)-12,13-epoxytrichothec-9-ene

A mixture of 4β,15-dihydroxy-3α-0-(2'-tetrahydropyranyl)-12,13-epoxytrichothec-9-ene (808 mg, 2.21 mmol), chloroacetic anhydride (1.132 g, 6.62 mmol) and pyridine (894 mg, 11.05 mmol) in 100 ml of CH₂ Cl₂ was stirred at room temperature for 14 h. The reaction mixture was diluted with 200 ml of CH₂ Cl₂ and washed with saturated NaHCO₃ solution, 1% HCl solution and brine. Drying over K₂ CO₃ and removal of the solvent gave 1.058 g (92%) of foam which was homogeneous on TLC. A portion of this material was purified by silica gel chromatography (elution with 0.5% methanol-CH₂ Cl₂) to furnish an analytical sample of title product. IR(KBr): 2955, 1762, 1740, 1290, 1186, 1172, 1129, 1080, 1039, 977 cm⁻¹.

B. 4β,15-Bis(chloroacetoxy)-3α-hydroxy-12,13-epoxytrichothec-9-ene

To a solution of 4β,15-bis(chloroacetoxy)-3α-0-(2'-tetrahydropyranyl)-12,13-epoxytrichothec-9-ene (858 mg, 1.65 mmol) in 100 ml of 95% ethanol was added 19 ml of 1 N HCl solution. The resulting solution was stirred at room temperature for 24 hours. The reaction mixture was diluted with CH₂ Cl₂ (300 ml) and washed with saturated NaHCO₃ solution and brine. Drying over K₂ CO₃ -Na₂ SO₄ and removal of the solvent gave 600 mg of foam. Chromatography of this material on silica gel (elution with 1% methanol-CH₂ Cl₂) gave 524 mg (73%) of 4β,15-bis(chloroacetoxy)-3α-hydroxy-12,13-epoxytrichothec-9-ene. An analytical sample was obtained by recrystallization from chloroform-diethyl ether, m.p. 139°-141° C. IR(KBr): 3450, 2970, 2913, 1758, 1742, 1327, 1293, 1192, 1173, 1083, 1008, 967 cm⁻¹.

Anal. Calc'd. for C₁₉ H₂₄ O₇ Cl₂ : C, 52.42; H, 5.56. Found: C, 52.31; H, 5.34.

Elution of the silica gel column with 2% methanol-CH₂ Cl₂ gave 110 mg (15%) of 15-chloroacetoxy-3α,4β-dihydroxy-12,13-epoxytrichothec-9-ene.

EXAMPLE 3 15-Acetoxy-4β-chloroacetoxy-3α-hydroxy-12,13-epoxytrichothec-9-ene

A mixture of 15-acetoxy-4β-hydroxy-3α-0-(2'-tetrahydropyranyl)-12,13-epoxytrichothec-9-ene (785 mg, 1.92 mmol), chloroacetic anhydride (492 mg, 2.88 mmol) and pyridine (0.309 ml, 3.84 mmol) in 100 ml of CH₂ Cl₂ was stirred at room temperature for 28.5 h. The reaction mixture was diluted with CH₂ Cl₂ (200 ml) and washed with saturated NaHCO₃ solution and brine. Drying over K₂ CO₃ and removal of the solvent gave 931 mg (100%) of a white foam. The NMR and IR spectra of this material were consistent with the structure of 15-acetoxy-4β-chloroacetoxy-3α-0-(2'-tetrahydropyranyl)-12,13-epoxytrichothec-9-ene.

To a solution of 15-acetoxy-4β-chloroacetoxy-3α-0-(2'-tetrahydropyranyl)-12,13-epoxytrichothec-9-ene (900 mg, 1.98 mmol) in 100 ml of 95% ethanol was added 19 ml of 1 N HCl solution. After 10 h of stirring at room temperature, 200 ml of CH₂ Cl₂ was added to the reaction mixture. The aqueous layer which separated was extracted with 25 ml of CH₂ Cl₂. The combined CH₂ Cl₂ layers were washed with saturated NaHCO₃ solution and brine. Drying over K₂ CO₃ -Na₂ SO₄ and removal of the solvent gave 662 mg of foam. Chromatography on silica gel (elution with 0.5% methanol-CH₂ Cl₂) gave 350 mg (44%) of the title compound. Recrystallization from diethyl ether furnished the analytical sample; m.p. 166°-167.5° C. IR(KBr): 3500, 3040, 3020, 2989, 2918, 1754, 1736, 1378, 1330, 1260, 1208, 1167, 1074, 1052, 960 cm.sup. -1.

Anal. Calc'd for C₁₉ H₂₅ O₇ Cl: C, 56.83; H, 6.29. Found: C, 57.12; H, 6.29.

EXAMPLE 4 15-(trans-2'-Butenoyloxy)-3α,4β-dihydroxy-12,13-epoxytrichothec-9-ene

A solution containing 366 mg (1 mmol) of 3α-0-(2'-tetrahydropyranyl)-4β,15-dihydroxy-12,13-epoxytrichothec-9-ene and 395 mg (5 mmol) of dry pyridine in 50 ml of CH₂ Cl₂ (previously dried over 4 A molecular sieves) was cooled at 5° C. To the stirred solution was added dropwise 261 mg (2.5 mmol) of trans-2-butenoic acid chloride and, after completion of the addition, the mixture was stirred for 1 h at 5° C. and for 16 h at ambient temperature. The solution was diluted with 50 ml of CH₂ Cl₂ and was successively washed with saturated aqueous NaHCO₃, brine, 1% aqueous HCl and brine. The organic phase was dried over Na₂ SO₄ and the solvent was evaporated under reduced pressure to provide 360 mg of a gum. This was dissolved in 50 ml of 95% ethanol and to it was added 5 ml of 2 N HCl. After the solution had been stored at room temperature for 22 h, it was diluted with 100 ml of CH₂ Cl₂ and was washed successively with H₂ O, saturated aqueous NaHCO₃ and brine. The organic phase was dried over Na₂ SO₄ and the solvent was evaporated under reduced pressure to provide 260 mg of gum. This was chromatographed on 20 g of silica gel using 1% methanol in CH₂ Cl₂ as the solvent. The first product eluted was 4β,15-bis-(trans-2'-butenoyloxy)-3α-hydroxy-12,13-epoxytrichothec-9-ene (26 mg) followed by 22 mg of 4β(trans-2'-butenoyloxy)-3α,15-dihydroxy-12,13-epoxytrichothec-9-ene and then 147 mg of the title compound which was crystallized from CHCl₃ -SKELLYSOLVE B as a white solid of mp 83°-86°. IR(KBr): 3440, 2970, 1725, 1190, 1110, 1085, 965 cm⁻¹.

Anal. Calc'd for C₁₉ H₂₆ O₆.0.5H₂ O: C, 63.49; H, 7.57. Found: C, 63.54; H, 7.43.

EXAMPLE 5 4β,15-Bis-(trans-2'-butenoyloxy)-3α-hydroxy-12,13-epoxytrichothec-9-ene

Repetition of the above experiment using 6 equivalents of trans-2-butenoic acid chloride gave the title compound as a hygroscopic white foam. IR(KBr): 3420, 2970, 1720, 1310, 1260, 1185, 965 cm⁻¹. In addition, there was obtained 4β(trans-2'-butenoyloxy)-3α,15-dihydroxy-12,13-epoxytrichothec-9-ene as a cream solid of m.p. 60°-62° C. IR(KBr): 3460, 2960, 1710, 1315, 1190, 1105, 1080, 955 cm⁻¹.

Anal. Calcd for C₁₉ H₂₆ O₆.0.25H₂ O: C, 64.30; H, 7.53. Found: C, 64.19; H, 8.06.

EXAMPLE 6 15-(2'-Methylpropenoyloxy)-3α,4β-dihydroxy-12,13-epoxytrichothec-9-ene

To a solution of 366 mg (1 mmol) of 3α-0-(2'-tetrahydropyranyl)-4β,15-dihydroxy-12,13-epoxytrichothec-9-ene and 395 mg (5 mmol) of dry pyridine in 50 ml of methylene chloride (dried over 4 A molecular sieves) was added with stirring 261 mg (2.5 mmol) of freshly distilled 2-methylpropenoic acid chloride. The solution was stored at 22° C. for 17 h and was then treated with an additional 261 mg (2.5 mmol) of the acid chloride. After a further 22 h at 22° C., the solution was worked up as described in Example 4 and the residue was chromatographed on 20 g of silica gel. 2-Methylpropenoic acid anhydride was eluted using 1% methanol in CH₂ Cl₂. The solvent was changed to methanol to elute 230 mg of white foam which was hydrolyzed as described above (Example 4) to give 189 mg of a foam. This was chromatographed on 20 g silica gel using 1% methanol in CH₂ Cl₂ as the solvent. Minor products were eluted and the solvent was changed to 20% methanol in CH₂ Cl₂ to afford 116 mg (33%) of the title compound as a foam which crystallized from CH₂ Cl₂ -SKELLYSOLVE B as a pale pink solid of m.p. 79°-81° C. IR(KBr): 3440, 2960, 1715, 1165, 1080, 955 cm⁻¹.

Anal. Calc'd for C₁₉ H₂₆ O₆.0.5H₂ O: C, 63.49; H, 7.57. Found: C, 63.36; H, 7.40.

EXAMPLE 7 4β-(Chloroacetoxy)-15-(2'-methylpropenoyloxy)-3α-hydroxy-12,13-epoxytrichothec-9-ene A. 3α-0-(2'-Tetrahydropyranyl)-15-(2'-methylpropenoyloxy)-4β-hydroxy-12,13-epoxytrichothec-9-ene

A solution containing 3.66 g (0.01 mol) of 3α-0-(2'-tetrahydropyranyl)-4β,15-dihydroxy-12,13-epoxytrichothec-9-ene, 3.95 g (0.05 mol) of pyridine and 2.61 g (0.025 mol) of freshly distilled 2-methylpropenoic acid chloride in 250 ml of dry methylene chloride was stirred for 16 h at 22° C. An additional 2.61 g (0.025 mol) of the acid chloride was added and stirring was continued for 6 h. The solution was diluted with CH₂ Cl₂ and was washed in succession with saturated aqueous NaHCO₃, brine, 1% aqueous HCl and brine. The organic phase was dried over Na₂ SO₄ and the solvent evaporated under reduced pressure to give 5.36 g of an oil. This was chromatographed on 100 g of silica gel using 1% methanol in CH₂ Cl₂ as the solvent. 2-Methylpropenoic acid anhydride was first eluted, followed by 615 mg of a foam which was hydrolysed as before (Example 4) in 67.5 ml of 95% ethanol and 13.5 ml of 1 N HCl. The usual work-up gave 590 mg of gum from which, by chromatography, 198 mg of 4β,15-bis-(2'-methylpropenoyloxy)-3α-hydroxy-12,13-epoxytrichothec-9-ene was isolated as a hygroscopic foam IR(KBr): 3500, 2960, 1720, 1165, 1080, 960 cm⁻¹ ; which was identified by its NMR spectrum. The next fraction from this chromatography afforded 4β-(2'-methylpropenoyloxy)-3α,15-dihydroxy-12,13-epoxytrichothec-9-ene as colorless crystals of m.p. 175°-176° C. IR(KBr): 3510, 3460, 2500, 1690, 1330, 1300, 1170, 1080, 1060, 910, 900 cm⁻¹.

Anal. Calc'd for C₁₉ H₂₆ O₆.0.25H₂ O: C, 64.30; H, 7.53. Found: C, 64.24; H, 7.14.

From the chromatographic separation of the tetrahydropyranyl ethers (above) there was next obtained 810 mg of a foam which was re-chromatographed on fresh silica gel (20 g) using the same solvent system to provide 3α-0-(2'-tetrahydropyranyl)-15-(2'-methylpropenoyloxy)-4β-hydroxy-12,13-epoxytrichothec-9-ene as a foam.

B. 4β-(Chloroacetoxy)-15-(2'-methylpropenoyloxy)-3α-hydroxy-12,13-epoxytrichothec-9-ene

To a stirred solution of 164 mg (0.38 mmol) of 3α-0-(2'-tetrahydropyranyl)-15-(2'-methylpropenoyloxy)-4β-hydroxy-12,13-epoxytrichothec-9-ene in 25 ml of dry CH₂ Cl₂ were added in succession 36 mg (0.46 mmol) of pyridine and 78 mg (0.46 mmol) of chloroacetic anhydride. The solution was stored for 17 h at 22° C. The solution was worked up as before and hydrolysed as usual with 27 ml of 95% ethanol and 5.4 ml of 1 N HCl. After work-up as before there was obtained a gum which was triturated with SKELLYSOLVE B to provide a hydroscopic solid of m.p. 58°-60° C. IR(KBr): 2960, 1755, 1715, 1320, 1295, 1165, 1085, 955 cm⁻¹.

Anal. Calc'd for C₂₁ H₂₇ ClO₇ : C, 59.08; H, 6.38. Found: C, 60.48; H, 6.66.

EXAMPLE 8 15-(2'-Chloropropionyloxy)-3α,4β-dihydroxy-12,13-epoxytrichothec-9-ene ##STR40##

To a mixture of scirpentriol (1.12 g) in 2,6-lutidine (10 ml), there was added with cooling 1 ml of 2-chloropropionyl chloride. After 24 hours at room temperature the solution was poured on ice, and the mixture was extracted with 3×15 ml of ethyl acetate. The organic layer was washed with water and dilute sodium bicarbonate. After evaporation of the dried solvent, there remained 1.1 g of an oil. This was dissolved in 8 ml of benzene and placed on a silica gel column (Grace, 1.5×49 cm). The column was eluted with benzene containing increasing proportions of methanol. The title compound emerged with 2.5% methanol and appeared homogeneous on TLC plates (SiO₂) developed with ethyl acetate-toluene (3:1 v/v). Yield: 28.8 mg.

The NMR spectrum (CDCl₃, 100 MHz) of the product showed the following peaks:

    ______________________________________                                         ppm                                                                            ______________________________________                                         0.7 (s, 3H)      C-14                                                          1.7-2.0 (m, 10H) C-16, C-7, C-8, CH.sub.3 CHCl                                 2.8-3.1 (dd, 2H) C-13                                                          3.7 (d, 1H)      C-2                                                           3.9-4.1 (dd, 2H) C-15                                                          4.3-4.5 (m, 3H)  C-3, C-4, C-11                                                4.4-4.6 (q, 1H)  CHClCH.sub.3                                                  5.5 (d, 1H)      C-10                                                          ______________________________________                                    

NMR showed that C-15 was acylated (C-15 protons appeared at 3.9-4.1 ppm); protons at C-3 and C-4 appeared approximately at 4.5 ppm indicating that they were not acylated.

EXAMPLE 9 15-Benzoyloxy-3α,4β-dihydroxy-12,13-epoxytrichothec-9-ene ##STR41##

3α,4β,15-Tribenzoyl-12,13-epoxytrichothec-9-ene (1.4 g) was hydrolyzed for 4.5 hours in a solution of 230 ml of methanol, 19 ml of concentrated ammonium hydroxide and 31 ml of water. The mixture was concentrated and extracted with benzene (3×50 ml). The volume was reduced to 25-30 ml which was placed on a silica gel column (Grace, 2×49 cm) and eluted with benzene containing increments of methanol. The fractions were monitored on TLC plates (SiO₂) with benzene-methanol (188:12 v/v) as developer. Spots were visualized with an alkaline permanganate spray. The fraction emerging at 2% methanol yielded 56 mg of the pure 4β,15-dibenzoate; at 3% methanol 221 mg of the title compound was obtained.

NMR (CDCl₃, 100 MHz) showed the following peaks:

    ______________________________________                                         ppm                                                                            ______________________________________                                         0.8 (s, 3H)         C-14                                                       1.7 (s, 3H)         C-16                                                       2.0 (m, 4H)         C-7, C-8                                                   2.7-3.0 (dd, 2H)    C-13                                                       3.9-4.3 (m, 3H)     C-3, C-4, C-11                                             4.0-4.4 (m, 2H)     C-15                                                       5.5 (d, 1H)         C-10                                                       7.1-8 (m, 5H)       benzoyl                                                    ______________________________________                                    

C-3 and C-4 protons appeared at 3.9-4.3 ppm indicating that they were not acylated.

The starting material, 3α,4β,15-tribenzoyloxy-12,13-epoxytrichothec-9-ene, in this experiment was prepared by acylating scirpentriol with benzoyl chloride according to the following procedure:

To scirpentriol (840 mg) dissolved in 6 ml of pyridine, there was added 1.3 g of benzoyl chloride. After 24 hours at room temperature the solution was added to ice. The usual work-up gave a solid which was crystallized from ether-hexane to give 350 mg of 3α,4β,15-tribenzoyloxy-12,13-epoxytrichothec-9,10-ene.

The NMR spectrum (CDCl₃, 100 MHz) of the product showed the following peaks:

    ______________________________________                                         ppm                                                                            ______________________________________                                         1.05 (s, 3H)          C-14                                                     1.75 (s, 3H)          C-16                                                     2.10 (m, 4H)          C-7, C-8                                                 2.95-3.25 (dd, 2H)    C-13                                                     4.2 (d, 1H)           C-11                                                     4.5-4.8 (m, 2H)       C-15                                                     5.5-5.6 (d, 1H)       C-10                                                     5.7 (m, 1H)           C-3                                                      6.2 (d, 1H)           C-4                                                      7.3-8.2 (m, 15H)      (benzoyl)                                                ______________________________________                                    

The compound contains 3 benzoyl groups, protons at C-3, C-4 and C-15 are on carbons bearing acylated hydroxyl groups.

EXAMPLE 10 9,10β-Epoxy-15-(trans-2'-butenoyloxy)-3α,4β-dihydroxy-12,13-epoxytrichothec-9-ene ##STR42##

A solution containing approximately equimolar amounts of the bis-ester of Example 5 and m-chloroperbenzoic acid in CH₂ Cl₂ was stirred at room temperature for 72 hours. The resulting solution was washed in succession with 10% aqueous Na₂ SO₃, saturated aqueous NaHCO₃ and brine. The organic phase was dried over Na₂ SO₄ and the solvent was evaporated under reduced pressure to give the title product, m.p. 83°-85° C.

Anal. Calcd. for C₁₉ H₂₆ O₇ 0.5H₂ O: C, 60.78; H, 7.25. Found: C, 60.52; H, 7.25. 

We claim:
 1. The compound having the formula ##STR43## 